Therapeutic glycosylated molecules intended for use in humans should have complex glycosylation patterns similar to those found in humans. Therefore, animal cells are generally used to produce therapeutic glycosylated molecules, such as proteins, or lipids, where it is desirable that the glycosylated molecules have a complex, human-like glycosylation pattern. The structure and complexity of glycans severely affects in vivo function of the biomolecule via modulation of half life, receptor binding, induction or suppression of immune reactions.
Sugar chains of glycolipids are complex and can contain a significant amount of Fucose (PNAS 1985; 82: 3045-3049.). Sugar chains of glycoproteins are roughly divided into two types, namely sugar chains which bind to asparagine (N-glycoside-linked sugar chain) and sugar chains which bind to other amino acid such as serine, threonine (O-glycoside-linked sugar chain), based on the binding form to the protein moiety.
N-glycoside-linked sugar chains have various structures (Biochemical Experimentation Method 23-Method for Studying Glycoprotein Sugar Chain (Gakujutsu Shuppan Center), edited by Reiko Takahashi (1989)), but it is known that they have a basic common core structure. The sugar chain terminus which binds to asparagine is called a reducing end, and the opposite side is called a non-reducing end. N-glycoside-linked sugar chain includes a high mannose type in which mannose alone binds to the non-reducing end of the core structure; a complex type in which the non-reducing end side of the trimannose core typically has at least one galactose-N-acetylglucosamine (hereinafter referred to as Gal-GlcNAc) attached to each of the two (1,3 and 1,6) mannose arms The non-reducing end side of Gal-GlcNAc may contain galactose and sialic acid, bisecting N-acetylglucosamine or the like. In a hybrid type the non-reducing end side of the core structure has branches of both of the high mannose type and complex type. In glycans from vertebrate cells fucose may be attached to the antennary GlcNAc via an alpha 1,3 linkage (terminal fucose) or to the asparagine-linked GlcNAc via an alpha 1,6 linkage (core fucose). Insect cells produce glycans which may contain 1,3 linked core fucose.
The oligosaccharide moiety of N-glycosylated proteins is initially biosynthesized from lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol which is then transferred to asparagine occurring in the tripeptide sequence Asn-X-Ser or Thr, where X could be any amino acid except Pro, of a protein in the endoplasmic reticulum (ER). Afterwards, the protein is transported to the Golgi-apparatus, where the oligosaccharide portion is further processed in the following sequence: First, all three glucose (Glc) residues are removed by glucosidases I and II to yield Man9GlcNAc2-protein. The Man9GlcNAc2 structure may be further processed by the removal of a number of mannose (Man) residues. Initially, four α-1,2-linked mannoses are removed to give a Man5GlcNAc2-protein which is then lengthened by the addition of a N-acetylglucosamine (GlcNAc) residue. This new structure, the GlcNAcMan5GlcNAc2-protein, is the substrate for mannosidase Il which removes the α-1,3- and α-1,6-linked mannoses. Thereafter, the other sugars, GlcNAc, galactose, fucose and sialic acid, are added sequentially to give the complex types of structures often found on N-glycosylated proteins.
An IgG molecule, for example, contains a N-linked oligosaccharide covalently attached at the conserved Asn297 of each of the CH2 domains in the Fc region. The oligosaccharides found in the Fc region of serum IgGs are mostly biantennary glycans of the complex type. Variations of IgG glycosylation patterns include the attachment of terminal sialic acid (NeuAc), a third GlcNac arm (bisecting GlcNAc), a terminal galactosylation (G), and α-1,6-linked core fucosylation (F) to the core structure: 2× N-Acetylglucosamin (GlcNAc) and 3× mannose (Man) (GlcNAc2Man3). The exact pattern of glycosylation depends on the structural properties of IgG subcomponents, in particular, CH2 and CH3 domains (Lund et al. (2000) Eur. J. Biochem., 267: 7246-7257).
Animal and human cells have fucosyltransferases that add a fucose residue to the GlcNAc residue at the reducing end of the N-glycans on a protein or to other nascent glycostructures on glycolipids. Fucosylation of protein- or lipid-bound glycomoieties requires a nucleotide sugar, GDP-L-fucose, as a donor and also the presence of particular fucosyl transferases, which transfer the fucosyl residue from the donor to the acceptor molecule (Becker and Lowe, 1999). In eukaryotic cells GDP-L-fucose can be synthesized via two different pathways, either by the more prominent fucose de novo pathway or by the minor salvage pathway (Becker and Lowe, 1999). The salvage Pathway or “scavenger” pathway is a minor source of GDP-L-fucose (circa 10%) which can easily be blocked by omission of free fucose and fucosylated glycoproteins from the culture medium. The salvage pathway starts from extracellular Fucose which can be transported into the cytosolic compartment via fucose-specific plasma membrane transporters. Alternatively, fucose cleaved from endocytosed glycoproteins can enter the cytosol. Cytosolic L-fucose is phosphorylated by fucokinase to fucose-1-phosphate and then converted by GDP-Fucose Pyrophosphorylase to GDP-L-fucose (FIG. 1, right hand panel). Cell culture experiments suggest that the salvage pathway makes a relatively minor contribution to the cytosolic GDP-L-fucose pools (Becker and Lowe, 1999).
The more prominent fucose de novo pathway starts from GDP-D-mannose and consists of a GDP-mannose dehydratase (GMD) and GDP-keto-deoxy-mannose-epimerase/GDP-keto-deoxy-galactose-reductase (GMER, also known as Fx in humans), both located in the cytoplasm, which in concert converts GDP-mannose to GDP-L-fucose (FIG. 1, left hand panel). Later, GDP-L-fucose is transported into the Golgi via a GDP-fucose transporter located in the membrane of the Golgi apparatus. Once GDP-L-fucose has entered the Golgi luminal compartment, fucosyltransferases can covalently link GDP-L-fucose to nascent glycomoieties within the Golgi. In particular, Fucosyltransferase (Fut8) transfers the fucose residue by means of an 1,6-linkage to the 6 position of the GlcNAc residue at the reducing end of the N-glycan. The lack of fucose on glycoproteins has been shown to have specific advantages. For example, in monoclonal antibodies, immunoglobulins, and related molecules, it has been shown that absense of the core fucose sugar from the N-glycan attached to Asn297 of the Fc portion (CH2 domain) of immunoglobulins increases or alters its binding to Fc receptors. Different types of constant regions bind different Fc receptors. Examples include the binding of IgG1 Fc domains to cognate Fc receptors CD16 (FcγRIII), CD32 (FcγRII-B1 and -B2), or CD64 (FcγRI), the binding of IgA Fc domains to the cognate Fc receptor CD89 (FcαRI), and the binding of IgE domains to cognate Fc receptors FcεFR1 or CD23. Binding to the FcγRIII which is present on the surface of an NK Cells is strongly increased. (Shields et al. JBC 277 (30): 26733. (2002)).
A dominating mode of action of therapeutic antibodies is Antibody Dependent cytotoxicity (hereinafter referred to as “ADCC activity”). The antibody binding to a target cell (a tumor cell or a cell infected with a pathogen) with its Fab portion is recognised in its Fc portion by the Fc receptor of an effector cell, typically an NK cell. Once bound the effector cell releases cytokines such as IFN-γ, and cytotoxic granules containing perforin and granzymes that enter the target cell inducing cell death. The binding affinity to FcγRIII is critical for antibodies acting through ATCC. Carriers of a low affinity allele of the receptor respond poorly to therapeutic antibodies such as Rituximab (Cartron et al. Blood 99: 754-758).
Consequently, higher affinity to FcγRIII mediated by the absense of core fucose on the Fc glycan can increase the potency or reduce the effective dose of biotherapeutic product with major implications for clinical benefit and cost.
In order to modify the sugar chain structure of the produced glycoprotein, various methods have been attempted, such as 1) application of an inhibitor against an enzyme relating to the modification of a sugar chain, 2) homozygous knock out of a gene involved in sugar synthesis or transfer 3) selection of a mutant, 3) introduction of a gene encoding an enzyme relating to the modification of a sugar chain, and the like. Specific examples are described below.
Examples of inhibitors against enzymes relating to the modification of a sugar chains include castanospermin and N-methyl-1-deoxynojirimycin which are inhibitors of glycosidase I, bromocondulitol which is an inhibitor of glycosidase II, 1-deoxynojirimycin and 1,4-dioxy-1,4-imino-D-mannitol which are inhibitors of mannosidase I, swainsonine which is an inhibitor of mannosidase II and the like. Examples of an inhibitor specific for a glycosyltransferase include deoxy derivatives of substrates against N-acetylglucosamine transferase V (GnTV) and the like
Mutants of enzymes relating to the modification of sugar chains have been mainly selected and obtained from a lectin-resistant cell line. For example, CHO cell mutants have been obtained from a lectin-resistant cell line using a lectin such as WGA (wheat-germ agglutinin derived from T. vulgaris), ConA (cocanavalin A derived from C. ensiformis), RIC (a toxin derived from R. communis), L-PHA (leucoagglutinin derived from P. vulgaris), LCA (lentil agglutinin derived from L. culinaris), PSA (pea lectin derived from P. sativum) or the like.
Furthermore, several methods for producing recombinant antibodies lacking fucose have been reported. One of the most important enzymes that enable core fucosylation of N-glycomoieties is α-1,6-fucosyltransferase 8 (Fut8). Said enzyme catalyzes the binding of fucose to the 6-position of N-acetylglucosamine in the reducing end through an α-bond in a N-glycoside-linked sugar chain pentacore of a complex type N-glycan (WO 00/61739). Antibodies with reduced fucose content have also been achieved using a cell in which the expression of fucose transporter genes is artificially suppressed (US 20090061485). The introduction of RNA capable of suppressing the function of α-1,6-fucosyltransferase has also been described to lead to the production of antibody molecules lacking fucose (EP 1 792 987 A1).
Many of the proposed cells or methods for producing molecules having a modified glycosylation pattern, which can be used for therapeutic indications, have significant drawbacks. For example, the treatment of antibodies with enzymes that remove glycosylations, e.g. fucosidases to remove fucose residues, involves additional manufacturing steps with are expensive, time-consuming and which have potentially significant economic and drug consistency risks. Further, the molecular engineering of cell lines to knock-out key enzymes involved in the synthesis of glycoproteins is tedious, expensive and not always crowned with success. In addition, said cell lines have the disadvantage that they do not allow the “tunable” production of molecules with varying ADCC or CDC potency to optimize efficacy and safety for a therapeutic use. The treatment of cell lines with RNAi or antisense molecules to knock-down the level of key enzymes involved in the synthesis of glycoproteins can have unpredictable off-target effects, is costly and appears to be impractical for implementation at manufacturing-scale.
Thus, there is a need for novel advantageous cells and methods for the production of molecules with a modified glycosylation pattern having improved properties for therapeutic uses.
The present invention provides cells for the production of molecules having a modified glycosylation pattern, i.e. molecules which lack fucose, have a reduced amount of fucose or have other atypical sugars on their glycan structures. It also provides methods for the production of molecules having a modified glycosylation pattern, i.e. molecules which lack fucose, have a reduced amount of fucose or have other artificial sugars on their glycan structures, using said cells. Said molecules have improved properties for therapeutic uses, e.g. increased ADCC or CDC activity, enhanced ability to inhibit signalling events, increased ability to induce apoptosis, and/or increased ability for immune therapy. In addition, the cells and methods provided by the present invention allow the tunable, reliable, inexpensive and straight forward production of molecules having a modified glycosylation pattern, i.e. molecules lacking fucose, having a reduced amount of fucose or having other artificial sugars on their glycan structures. Furthermore, the present invention provides methods which are suitable for manufacturing scale-up.
In many cases, considerable time has been invested and huge efforts have been made to generate and develop effective producer cell lines that express the transgene of interest at desirable levels. In cases where the transgene of interest is a therapeutic antibody that could benefit from enhanced ADCC effector function, it would be desirable to further manipulate the producer cell line in such a way that the high producer cell is incapable of attaching core-fucose to the N-glycomoieties or is capable of attaching artificial sugars to the N-glycomoieties.
The present invention provides an expression unit which can easily be applied to already existing genetically engineered cells in a way that renders them incapable to attach fucose to nascent glycostructures of glycoproteins or that render them capable to attach other artificial sugars than fucose to nascent glycostructures of glycoproteins, e.g. in order to produce antibodies having an improved ADCC activity.